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Environmental fate & pathways

Bioaccumulation: aquatic / sediment

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Reference
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
Deviations:
no
Principles of method if other than guideline:
The study methods concerning the new chemical substance "The test on the degree of the bioconcentration in the bodies of fish and shellfish" (Kanpogyo-5, Yakuhatsu-615, 49-kikyoku-392,1974) (as revised partly on October 8, 1998)
GLP compliance:
yes
Radiolabelling:
no
Details on sampling:
Frequency of sampling: on Days 0, 7, 14, 21, 23 and 28 (exposure period) and on Days 3 and 7 (excretion period)

Sampling for fish:
Each six fish in the high and low exposure levels and two in the control group are sampled using landing net on analysis day. Putting them into ice water, the body surface is washed with tap water and wiped lightly by gauze. Then, the body weight is measured in a unit of 0.01 g using a balance (SOP/INS/4.2.2), and the body length from a mouth to a tail fin is measured in a unit of 0.1 cm. Two fish are used as one sample and analyzed in duplicate. Other sample is put into a plastic container respectively, and stored in a freezer after wrapped with aluminum-foil and packed in a plastic bag.
Fish are put into a homogenizer cup (SOP/INS/4.17) after mincing, and added 40 mL of acetonitrile. After homogenizing (8000 r.p.m., 8 min), the homogenate is transferred into a 250 mL centrifuge tube. The homogenizer cup is washed 2 times with 3 mL of acetonitrile for each time and the washing are also transferred into the centrifuge tube, and centrifuged for 10 min (8000 r.p.m., 10 °C). Then the supernatant is filtered through a glassfilter (No. GC50, Advantec Toyo Co., Ltd.) into a 50 mL volumetric flask.
Before filtration, a filter paper is washed with a little amount of acetonitrile. Washing the centrifugation tube with acetonitrile, and brought up to the mark with 50 mL of acetonitrile. Ten mL of the solution is taken into a round bottom flask with a pipet. The solution is concentrated to about 0.5 mL under a reduced pressure with a rotary evaporator in a water bath (25°C). The concentrated solution was transferred to a 1 mL volumetric flask. Then, 0.2 mL of distilled pure water was added to the volumetric flask and mixed well, and this solution was brought up to the mark with acetonitrile. An aliquot of this solution is filtered by an Ekikuro-disk 13CR and then the filtrate is analyzed by HPLC.

2 fish were used as one sample and the analysis was conducted at n = 2. In the control group, 2 fish were used as one sample and the analysis was conducted at n = 1.

Sampling for water:
Aliquots of test water (about 300 mL for the high exposure level and about 1 L for the low exposure level) are sampled from the central area of each aquarium with Erlenmeyer flasks.
After leaving, aliquots of the supernatant (200 mL for the high exposure level and 800 mL for the low exposure level) are measured with measuring cylinders. The measured test water of high exposure and low exposure level were brought up to 500 mL and 1 L, respectively in the separating funnel. The distilled pure water for washing of cylinders was added to the each funnel. And 100 mL of dichloromethane was added to each funnel. The solution is shaken with Shaker for 10 min. After standing, organic solvent layer (lower layer) for each exposure level group is collected in 200 mL of Erlenmeyer flask and each 20 g of anhydrous sodium sulfide is added and left for about 30 min. After dehydration, the solution is filtered through a glassfilter (No.GC 50, Advantech Toyo Co., Ltd.) into 200 mL of round bottom flask to remove anhydrous sulfide. In addition, the Erlenmeyer flask contained the
eluate and the residue are washed with 5 mL of dichloromethane for 5 times and collected into a round bottom flask. The filtrate is concentrated to dryness under a reduced pressure with a rotary evaporator (SOP/INS/4.14) in a water bath 25°C. The dried sample is dissolved in 1 mL of dichloromethane for the high exposure level and 0.2 mL of dichloromethane for the low exposure level. A small amount of methanol is added, the solution is transferred to 5 mL of to the round bottom flask for the high exposure level and 2 mL for the low exposure level. Then 1 mL and 0.4 mL of distilled pure water for washing the flask is added for the high exposure level and low exposure level. Furthermore, the sample is washed for several times with methanol, transferred into volumetric flask and brought up to the mark with methanol, after the solution is mixed completely and the temperature of the solution is constant. The sample solution is analyzed by HPLC.
Water analysis of control in the control group (at the time of study initiation) was conducted in the same manners that in low exposure level.
Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
50 mg of the test substance were weighed in an agate mortar and dissolved by adding dichloromethane. Furthermore, during diluting polyoxyethylenesorbitanmonoolate (Tween 80, 4 times of the test substance) in dichloromethane is added to the substance as a dispersant and mixed lightly.
After confirming that dichloromethane is volatilized, the resultant is brought up to 500 mL with distilled pure water with dissolving, and this solution is used as stock solution. After prepring the required quantity, the test solution was prepared by diluting the stock solution with water.

Control: the Tween 80 solution of 12 mg/L is diluted with water for study to obtain a concentration of 0.12 mg/L (TOC = 0.28 mg/L) for the test and loaded continuously into the test aquarium.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp (Lot No. 20424)
- Source: Kitamura Fish Farm (12-398 Guntiku, Yashiro-shi, Kumamoto)
- Length at study initiation (lenght definition, mean, range and SD): 5.7 +/- 0.6 cm
- Weight at study initiation (mean and range, SD): 1.89 +/- 0.29 g

ACCLIMATION
- Acclimation period: 7 days
- Acclimation conditions (same as test or not): flow through system at 25°C +/- 2°C
- Health during acclimation (any mortality observed): mortality was less than 5%
- Food type: mixed pelleted feed for carp (Swimmy, from Nippon Pet Food Company); crude protein: > 37.0% and crude lipid: > 2.5%
- Amount: ca. 2% of the body weight
- Frequency: daily, no feeding on the day before sampling
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
28 d
Total depuration duration:
7 d
Test temperature:
24.2 +/- 0.7°C
pH:
7.2 +/- 0.1
Dissolved oxygen:
7.5 +/- 0.1 mg/L
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: 100-L glass made (test water volume 50 L)
- Renewal rate of test solution (frequency/flow rate): 450 mL/min (648 L/day)
- No. of organisms per vessel: 48 for the high exposure level, 48 for the low exposure level and 10 fir the control containing the dispersant
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- dechlorinated public water of Saitama-shi
- Holding medium different from test medium: no

OTHER TEST CONDITIONS
- Light type and photoperiod: lightning for about 10 hours with fluorescent lights removed UV ray.

RANGE-FINDING / PRELIMINARY STUDY
- The 96h-LC50 of the test substance was determined to be 2.66 mg/L for Brachydanio rerio. Based on these results, the test concentrations were determined as follows:
High exposure level: 0.03 mg/L (+ 0.12 mg/L Tween 80)
Low exposure level: 0.003 mg/L (+ 0.012 mg/L Teen 80)
- Preliminary study:
Fish (Cyprinus carpio) were exposed at the high concentration level in semi-static system. On day 10 of exposure, fish (2 fish as one analysis) were sampled and analyzed. The bioconcentration factor of the test substance was found to be 146.
- The results of the preliminary study indicated that the test substance has a potential for bioaccumulation. The exposure (uptake) period and the excretion period were arranged to be for 28 days and 7 days, respectively.
Nominal and measured concentrations:
Nominal concentrations:
high exposure level: 0.03 mg/L (TOC = 0.76 mg/L)
low exposure level: 0.003 mg/L (TOC = 0.65 mg/L)
Measured concentrations:
See "Any other information on materials and methods incl. tables"
Reference substance (positive control):
no
Lipid content:
4.3 %
Time point:
start of exposure
Remarks on result:
other: based on the chloroform-methanol extraction
Lipid content:
4.6 %
Time point:
end of exposure
Remarks on result:
other: based on the chloroform-methanol extraction
Type:
BCF
Value:
758
Remarks on result:
other: BCF based on steady state and mean concentrations.
Remarks:
Conc.in environment / dose:0.03 mg/L
Type:
BCF
Value:
689
Remarks on result:
other: BCF based on steady state and mean concentrations
Remarks:
Conc.in environment / dose:0.003 mg/L
Details on results:
The bioconcentration factors of the test material at high and low exposure levels were 612-915 times and 536-905 times respectively. The bioconcentration under steady state at high and low exposure levels were 755 times and 684 times respectively.
The test substance was therefore found to be moderately bioaccumulating.
Both at exposure levels the steady state was brought at the day 7.

Excretion test
The mean values of residual rate of test material at day 7 were 12% and 4% in the high and low exposure levels respectively.
See "Any other information on results incl. tables"

Excretion test level:

Period of exposure High exposure level Low exposure level
  Sample no. (n = 2) Concentration of the test substance (μg/g) Residual rate (%) Sample no. (n = 2) Concentration of the test substance (μg/g) Residual rate (%)
3 1 6.57 28 1 0.326 16
2 4.47 19 2 0.184 9
7 1 3.35 14 1 0.129 6
2 2.59 11 2 < 0.0425 < 2
Validity criteria fulfilled:
yes
Conclusions:
At concentrations of 0.03 and 0.003 mg/l, the test substance was found to be midly bioaccumulative in the fish, the BCF being at about 758 and 689 respectively.
Executive summary:

In this guideline (OECD 305) study conducted to generally accepted scientific standards with GLP certification, the test material (EC 438-340-0) was determined to have a BCF of 758 and 689 (at respective concentrations of 0.03 and 0.003 mg/l). The test was conducted in Cyprinus carpio which were kept in natural fresh water under flow-through conditions. The test material was dissolved using dichloromethane with polyoxyethylenesorbitanmonoolate added as a dispersant. The use of a dispersant was considered not to affect the reliability of the study result.

Description of key information

Study conducted to generally recognised scientific standards with GLP. 

Key value for chemical safety assessment

BCF (aquatic species):
758 dimensionless

Additional information

In Article 13 of Regulation (EC) No 1907/2006, it is laid down that information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI (of the same Regulation) are met. Furthermore according to Article 25 of the same Regulation testing on vertebrate animals shall be undertaken only as a last resort.

  According to Annex XI of Regulation (EC) No 1907/2006 (Q)SAR results can be used if (1) the scientific validity of the (Q)SAR model has been established, (2) the substance falls within the applicability domain of the (Q)SAR model, (3) the results are adequate for the purpose of classification and labeling and/or risk assessment and (4) adequate and reliable documentation of the applied method is provided.

For the assessment of the test substance (Q)SAR results were used for aquatic bioaccumulation. The criteria listed in Annex XI of Regulation (EC) No 1907/2006 are considered to be adequately fulfilled and therefore the endpoint(s) sufficiently covered and suitable for risk assessment.

Therefore, and for reasons of animal welfare, further experimental studies on aquatic bioaccumulation are not provided.

 

A flow-through GLP guideline study according to OECD 305 has been conducted using Carp,Cyprinus carpio(Institute of Ecotoxicology Co., Ltd., Japan, 2002). Even though an emulsifier has been used the study is regarded as valid since both test concentrations (0.03 and 0.003 mg/L) are well below the water solubility of the test substance (1.9 – 2.8 mg/L). The exposure (uptake) period and the excretion period were arranged to be 28 days and 7 days, respectively. The study resulted in mean BCF values of 758 mg/L for the high exposure level (0.03 mg/L) and 689 mg/L for the low exposure level (0.003 mg/L) after 28 d of exposure. The excretion test was conducted subsequent to the exposure test. The mean values of the residual rate of the test material at day 7 were 12% and 4% in the high and low exposure level, respectively, showing excretion of the test substance. Therefore, the test substance is not bioaccumulative according to PBT criteria.

 

However, to support these findings four QSAR calculations have been conducted. The single models and their results are summarized in the table below:

 

Model

 

BCF

Log BCF

Remarks

Catalogic v5.11.17

 

407.38

2.61

all mitigating factors applied; 72.22 % in domain

T.E.S.T. v4.01

 

101.39

2.01

EPISuite v4.10

Regression-based estimate

61.2

1.79

The substance is within the applicability domain of the BCFBAF submodel: Bioconcentration factor

Arnot-Gobas upper trophic level

44.94

1.653

Including biotransformation rate estimates; MW and logKow are within the range of training set.

Arnot-Gobas mid trophic level

59.27

1.773

Including biotransformation rate estimates; MW and logKow are within the range of training set.

Arnot-Gobas lower trophic level

64.14

1.807

Including biotransformation rate estimates; MW and logKow are within the range of training set.

 VEGA CAESAR v2.1.13

 

10

 1.01

The test substance is out of model Applicability Domain

 

Regarding the results of the model calculation, the calculated BCF values range from 10 (VEGA) to 407.38 (Catalogic).

Of these models, Catalogic, VEGA CAESAR and the Arnot-Gobas model from EPISuite v4.10 take mitigating factors into account, e.g. metabolism, water solubility and/or size.

Catalogic revealed a corrected BCF of 407.38 and the compound is 72.22% within the model’s applicability domain.

The Arnot-Gobas model from the EPISuite takes into account the biotransformation rate of the compound and calculates BCF values for the upper, mid and lower trophic levels. The values for the present compound range from 44.94 (upper trophic level) to 64.14 (lower trophic level). The model assumes default lipid contents of 10.7%, 6.85% and 5.98% for the upper, middle and lower trophic levels, respectively. Usually, in the context of REACH a default lipid value of 5% is assumed which represents the average lipid content of the small fish used in OECD 305 studies. Thus, the higher lipid values of the Arnot-Gobas model can be regarded as reasonable worst-case scenarios as higher lipid contents are usually associated with a higher potential for bioaccumulation. The MW and the logKow are is within the range of training set and, therefore, the estimation is reliable.

The regression-based estimate from the EPISuite revealed a BCF value of 61.2 based on the measured logKow of 4.1 and the test substance is in the applicability domain of the model.

The T.E.S.T. package from the US EPA estimates BCF values using several different advanced QSAR methodologies. The recommended model of the T.E.S.T. package is the consensus method since it provides the most accurate prediction. This model estimates the BCF by taking an average of the predicted BCF values from the other applicable QSAR methods of the package. For the present substance the consensus method averaged the results from (1) the hierarchical clustering method, (2) the single model method, (3) the group contribution method, (4) the FDA method and (5) the nearest neighbor method. The resulting BCF value is 101.39.

The VEGA model was developed with several descriptors and is based on a dataset of 473 compounds. It offers detailed information on the applicability domain. In the present case, the calculation gave a BCF value of 10. Since the Global AD Index was calculated to be 0.234 the substance is out of the Applicability Domain of the model.

However, even though the test substance was out of the Applicability Domain of some programs, the QSAR results support the overall conclusion that the test substance is not bioaccumulative, since all QSAR models give a value far below a BCF of 2000 and the results of the models were the applicability domain is met are consistent with the results of all models. Furthermore, the results, where the applicability domain is met, are in line with the experimental BCF values.

In overall conclusion, considering the test results, the logPow of 4.1 and the QSAR calculations it can clearly be stated that test substance is not bioaccumulative according to PBT-criteria.